Goal of this project is to understand the mechanisms through which tyrosyl-tRNA synthetase (YRS) exerts its newly recognized role in regulating platelet production and, on the bases of this information, develop a novel therapeutic strategy for the treatment of thrombocytopenia. In preliminary studies we have determined that YRS exhibits the previously unrecognized property of accelerating megakaryocyte development and platelet generation. This effect appears to be independent of thrombopoietin (TPO) signaling because YRS can support the proliferation and maturation of c-Mpl-/- (TPO receptor knock-out) mouse megakaryocytes in vitro. The studies proposed in this application will establish the mechanisms underlying the novel function of YRS in the regulation of platelet production. We will also develop a pharmaceutical approach to a new class of drugs with the potential of enhancing platelet production in various diseases in which TPO mimetics alone are not effective. In aim 1, we will elucidate the molecular mechanisms by which YRS stimulates megakaryocytopoiesis. Based on preliminary studies, the effect of YRS on megakaryocytopoiesis is mediated by at least two mechanisms: (1) activation of monocytic cells and cytokine production, and (2) expansion of Sca1+CD11b+ progenitors that give rise to hematopoietic cells and vascular cells. We will identify the cells responsible to mediate the effect of YRS and the receptor(s) targeted in the process. Involvement of TLR-MyD88 pathway has been shown by preliminary testing of TLR2-/- and MyD88-/- mice. We will also focus on a unique population of Sca1+CD11b+ progenitor cells that are greatly expanded by YRS stimulation in bone marrow cell cultures. Our preliminary result suggested the differentiation potential of YRS-induced Sca1+CD11b+ progenitor cells into hematopoietic cells and vascular smooth muscle cells. We hypothesize that expansion of these progenitor cells also contributes to thrombocytopoiesis either by differentiating into hematopoietic progenitors or vascular cells that support proplatelet formation in the vascular niche. In aim 2, we will define the functional domains of YRS required to exert the effects on megakaryocytopoiesis, and use protein engineering to de- sign YRS variants with an optimal therapeutic window. In Aim 3, we will test the efficacy of engineered YRS as a potential drug for the treatment of thrombocytopenia using in vivo animal models. In addition to the acute immune-mediated thrombocytopenia model employed in preliminary studies, we are generating models of chronic thrombocytopenia induced by low-dose antibody administration, irradiation or chemotherapy to evaluate the effects of YRS in various conditions as may occur in human pathology. Finally, to test the possibility of thrombotic side effects, we will determine whether YRS affects platelet activation, platelet-leukocyte inter- actions and models of thrombosis. The studies proposed in this application will delineate a novel cellular pathway in which YRS contributes to the regulation of platelet production; and support the development of innovative approaches with translational potential for improving the treatment of thrombocytopenia.